Early deletion mutants of SV40 were studied in an attempt to determine those nucleotide sequences which affect the processing and stability of the two major early SV40 mRNAs. A mutant with a deletion located entirely within the coding region for large T-antigen generated a stable shortened large T-mRNA. In contrast, a deletion removing both large T and small t splice junctions as well as intervening sequences, produced no detectable stable early mRNA. The early SV 40 mutants with deletions between 0.53 and 0.60 map units (54/59 mutants) are viable in tissue culture infections. While all of these early viable mutants contain deletions in the intervening sequences for the large T-antigen, results from transcriptional mapping showed that none of these deletions appears to affect the sites or frequency of splicing for large T-mRNA. The 54/59 mutants in which the deletion removes the proximal small t splice junction make no discrete stable cytoplasmic small t-mRNA. Those 54/59 mutants with deletions that do not include the splice junction synthesize discrete and predictably shortened small t-mRNAs. The size and location of the specific deletions, however, appear to significantly affect the abundance of the altered transcripts; this probably reflects the frequency with which the splice is made. These findings suggest that both the primary sequence near the splice junctions and the secondary or tertiary RNA structure, influence the location and frequency with which a particular splicing event occurs. In addition, SDS-polyacrylamide gel analysis of the early SV40 polypeptides synthesized by the 54/59 mutants has suggested a direct relationship between the abundance of an mRNA and the amount of the corresponding polypeptide. These results suggest that the synthesis of the early SV40 proteins is regulated primarily by the synthesis and processing of a particular mRNA, rather than by translational or posttranslational controls.